[A comparative study on the properties of trichosanthin before and after site-directed PEGylation].
OBJECTIVE
To construct PEGylated trichosanthin (TCS) mutein and analyze its bioactivities, immunogenicity, acute toxicity, and pharmacokinetics.


METHODS
The potential antigenic determinant site YFF81-83 in the molecule of TCS was selected to undergo site-directed mutagenesis. Thus, a TCS mutein named TCS(YFF81-83ACS) was constructed and expressed in Escherichia coli of the line BL21 (DE3). Wild TCS (wTCS), TCSY(FF81-83ACS), and PEGylated TCS(YFF81-83ACS) (PEG- TCS(YFF81-83ACS)) of different concentrations were incubated with the supercoiled plasmid pUC19 to detect the DNAse activity, mixed with rabbit reticulocyte lysate to detect the ribosome inactivation activity, subcutaneously injected into 6 mice respectively to measure the serum IgG and IgE levels, intravenously injected into mice to observe the toxicity, and intravenously injected into SD rats to observe its -plasma half-life.


RESULTS
The DNAse activity of the PEG-TCS(YFF81-83ACS) was similar to that of the wTCS. The ribosome inactivation activity of the PEG-TCS(YFF81-83ACS) was 1/9-1/8 of that of the wTCS (P < 0.05). The serum IgE and IgG levels of the PEG-TCS(YFF81-83ACS) were both significantly lower than those of the wTCS (both P < 0.05). The LD50 of the PEG-TCS(YFF81-83ACS) was 1.8 times that of the wTCS (P < 0.05). The mean residence time and plasma half-life of the PEG-TCS(YFF81-83ACS) were significantly increased and its plasma clearance was significantly decreased (all P < 0.05).


CONCLUSIONS
Site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.
An
Qun-xing
QX
Department of Blood Transfusion, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Lei
Ying-feng
YF
Mu
Shi-jie
SJ
Zhang
Xian-qing
XQ
Chen
Rui
R
Xia
Ai-jun
AJ
Chen
Chen
C
Yi
Jing
J
Wu
Yuan-ru
YR
Yu
Rui
R
Xu
Zhi-kai
ZK
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000276:immunology* / Q000493:pharmacokinetics / Q000633:toxicity*; D017354:Point Mutation; D011092:Polyethylene Glycols / Q000737:chemistry*; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D023382:Toxicity Tests, Acute; D015978:Trichosanthin / Q000097:blood / Q000737:chemistry / Q000235:genetics*
D050505:Mutant Proteins; D011092:Polyethylene Glycols; D015978:Trichosanthin